Human neurokinin-1 receptor

ABSTRACT

Recombinant human neurokinin-1 receptor (hereinafter identified as human NK1R) has been prepared by polymerase chain reaction techniques. The complete sequence of human NK1R complementary DNA has been identified. Expression systems, including a CHO (chinese hamster ovarian cell line) stable expression system; and an assay using the CHO expression system have been developed. NK1R, also known as substance P receptor, may be used in an assay to identify and evaluate entities that bind to the substance P receptor. The assay may also be used in conjunction with diagnosis and therapy to determine the body fluid concentration of substance P antagonists in arthritis patients.

This is a continuation of application Ser. No. 07/691,197, filed on Apr.25, 1991, now abandoned.

BACKGROUND OF THE INVENTION

The present invention concerns cloned human neurokinin-1 receptor (humanNK1R) and recombinant human NK1R. Neurokinin-1 receptor is also known assubstance P receptor.

J. Yokota, et al., J. Biol. Chem., 264:17649 (1989) have reported clonedrat neurokinin-1 receptor. N. P. Gerard, et al., J. Biol. Chem.,265:20455 (1990), have reported human neurokinin-2 receptor. Cloned ratand bovine neurokinin-2 receptor have likewise been reported. Seerespectively, Y. Sasi, and S. Nakanishi, Biochem Biophys. Res. Comm.,165:695 (1989), and Y. Masu, et al., Nature 329:836 (1987). Cloned ratneurokinin-3 receptor has also been reported by R. Shigemoto, et al., J.Biol. Chem., 265:623 (1990).

The above references, however, neither disclose or suggest the instantinvention. In particular, the pharmacological profile of the humanreceptor differs significantly from the rat. Moreover, the ratneurokinin-1 receptor differs from the NK1R disclosed herein by 23 aminoacids.

Substance P is a naturally occuring undecapeptide belonging to thetachykinin family of peptides. Substance P is a pharmacologically-activeneuropeptide that is produced in mammals. Its characteristic amino acidsequence is illustrated in U.S. Pat. No. 4,680,283. As is well known inthe art substance P and other tachykinins have been implicated in thepathophysiology of numerous diseases. Substance P has been shown to beinvolved in the transmission of pain or migraine (see B. E. B. Sandberget al., Journal of Medicinal Chemistry, Vol. 25, p. 1009 (1982)), aswell as in central nervous system disorders such as anxiety andschizophrenia, in respiratory and inflammatory diseases such as asthmaand rheumatoid arthritis, respectively, and in gastrointestinaldisorders and diseases of the GI tract, like ulcerative colitis andCrohn's disease, etc. (see D. Regoli in "Trends in Cluster Headache,"edited by F. Sicuteri et al., Elsevier Scientific Publishers, Amsterdam,1987, pp. 85-95).

The instant invention also concerns an assay protocol which can be usedto determine substance P activity in body fluids. The assay can also beused for identifying and evaluating substances that bind substance Preceptor. Thus, the assay can be used to identify substance Pantagonists and evaluate their binding affinity. Other methods includesthat described by M. A. Cascieri, et al., J. Biol. Chem., 258-5158(1983).

By use of such methods, substance P antagonists have been identified.See, for example, R. M. Snider, et al., Science, 251:435 (January 1991)and S. McLean, et al., Science, 251:437 (January 1991). See alsoWO90/05525 which published May 31, 1990, which is hereby incorporated byreference. Methods to date have proven inferior, in part, for failure ofthe animal receptor (animal NK1R, NK2R or NK3R) activity to accuratelyreflect that of human neurokinin-1 receptor. Furthermore, prior to thisdisclosure human NK1R has not been available in a purified form or insubstantial isolation from NK2R and/or NK3R.

Use of such neurokinin receptor sources can not accurately depict theaffinity for human NK1R.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Full length amino acid sequence of human neurokinin-1 receptor.

FIG. 2 Full length nucleotide sequence of the

cloned human neurokinin-1 receptor complementary DNA.

FIG. 3 Competitive binding of substance P (SP), substance K (SK) andhuman neurokinin-1 receptor (NK1R) in COS assay.

SUMMARY OF THE INVENTION

Recombinant human neurokinin-1 receptor (human NK1R) is disclosed whichhas been prepared by polymerase chain reaction techniques. Alsodisclosed is the complete sequence of human NK1R complementary DNA;expression systems, including a CHO (chinese hamster ovarian cell line)stable expression system; and an assay using the CHO expression system.

NK1R, also known as substance P receptor, can be used in an assay toidentify and evaluate entities that bind substance P receptor. The assaycan also be used in conjunction with diagnosis and therapy to determinethe body fluid concentration of substance P in arthritis patients.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention concerns human neurokinin-1 receptor,said receptor being free of other human receptor proteins.

In one class this embodiment concerns human neurokinin-1 receptor, saidreceptor being free of other human proteins.

Within this class, this embodiment concerns human neurokinin-1 receptorfrom human cells such as glioblastoma, said receptor being free of otherproteins.

In a second class, this embodiment concerns a protein comprising the 407amino acid sequence depicted in FIG. 1, said protein being free of otherhuman receptor proteins.

Within the second class this embodiment concerns a protein consisting ofthe 407 amino acid sequence as shown in FIG. 1.

The first embodiment also concerns a pharmaceutical composition forinhibiting the binding of substance P to cellular neueokinin-1 receptor,said composition comprising an effective amount of neurokinin-1receptor.

The first embodiment also concerns a method of inhibiting the binding ofsubstance P to cellular human neurokinin-1 receptor, in a patient inneed of such inhibition, comprising: administration of an effectiveamount of human neurokinin-1 receptor.

The use of such pharmaceutical compositions and methods for antagonisingthe binding of substance P to in vivo neurokinin-1 receptor is disclosedin, for example, R. M. Snider, et al., Science, 251:435 (January 1991);S. McLean, et al., Science, 251:437 (January 1991); and WO90/05525 whichpublished May 31, 1990, which are hereby incorporated by reference.:

A second embodiment concerns a DNA sequence encoding human neurokininreceptor complementary DNA, said DNA, said sequence being free of otherhuman DNA sequences.

As will be appreciated by those of skill in the art, there is asubstantial amount of redundancy in the set of condons which translatespecific amino acids. Accordingly, the invention also includesalternative base sequences wherein a codon (or codons) are replaced withanother codon, such that the amino acid sequence translated by the DNAsequence remains unchanged. For purposes of this specification, asequence bearing one or more such replaced codons will be defined as adegenetate variation. Also included are mutations (exchange ofindividual amino acids) which one of skill in the art would expect tohave no effect on functionality, such as valine for leucine, argininefor lysine and asparigine for glutamine.

One class of the second embodiment the invention concerns the nucleotidesequence of complementary DNA, beginning with nucleotide 123 and endingwith necleotide 1346 as shown in FIG. 2.

Within this class of the second embodiment is the DNA sequence (SEQ IDNO:1) that further comprises: ##STR1## or a degenerate variationthereof.

The second embodiment the invention concerns the partial nucleotidesequence of complementary DNA, as shown in FIG. 2 or a degeneratevariation thereof.

A third embodiment of this invention concerns systems for expressinghuman neurokinin receptor.

One class this third embodiment of the invention comprises:

A plasmid which comprises:

(a) a mammalian expression vector, such as pRcCMV, and

(b) a base sequence encoding human neurokinin-1 receptor protein.

Within this class of the third embodiment the neurokinin-1 receptorcomprises the nucleotide sequence of complementary DNA, beginning withnucleotide 123 and ending with necleotide 1346 as shown in FIG. 2.

A second class of this third embodiment of the invention concerns asystem for the transient expression of human neurokinin-1 receptor(NK1R) in a monkey kidney cell line (COS).

A third class of this third embodiment of the invention concerns asystem for the expression of human neurokinin-1 receptor in a chinesehamster ovarian cell line (CHO), the system comprising a vectorcomprising human neurokinin receptor (NK1R) cDNA.

Within this class of the third embodiment is is the sub-class whereinthe expression system includes

A plasmid which comprises:

(a) a mammalian expression vector, such as pRcCMV, and

(b) a base sequence encoding human neurokinin-1 receptor protein.

Within this sub-class the neurokinin-1 receptor comprises the nucleotidesequence of complementary DNA, beginning with nucleotide 123 and endingwith necleotide 1346 as shown in FIG. 2. is subclosed into the vectorpRcCMV.

A forth embodiment of the invention concerns a method of using any ofthe above expression systems for determining the binding affinity of atest sample for human neurokinin-1 receptor.

In one class this embodiment concerns a method of using a Chinesehamster ovarian cell line, said line transplanted with a plasmid, whichplasmid comprises:

(a) vector pRcCMV, and

(b) the base sequence encoding human neurokinin-1 receptor protein, themethod which comprises:

(1) expressing human neurokinin-1 receptor in said CHO cells;

(2) addition of a test sample to a solution containing ¹²⁵ I-substance pand said cells;

(3) incubating the products of Step (1), wherein said incubationeffective for expressing said the human neurokinin-1 receptor andeffective for competitive binding of said ¹²⁵ I-substance P and saidtest sample to said human neurokinin-1 receptor;

(4) separating said ¹²⁵ I-substance P which is bound to said humanneurokinin-1 receptor from said ¹²⁵ I-substance P which is not bound;

(5) measuring the radioactivity of said ¹²⁵ I-substance P which is boundto said human neurokinin-1 receptor.

In a second class this embodiment concerns a method of using a Chinesehamster ovarian cell line (CHO), said line transplanted with a plasmidwhich plasmid comprises

(a) vector pRcCMV, and

(b) the base sequence encoding human neurokinin-1 receptor protein, themethod comprising:

(1) expressing human neurokinin-1 receptor in said CHO cells;

(2) equilibrating the product of Step (1) with ³ H-myoinositol;

(3) washing the product of Step (2);

(4) incubating the product of Step (3) with a test sample in thepresence of 10 mM LiCl, which results in the production of inositolmonophosphate;

(5) measuring the inositol monophosphate.

In overview, the present invention describes methods to isolate thehuman neurokinin-1 receptor (human NK1R) complementary DNA (cDNA)without prior knowledge of its protein sequence or gene sequence. HumanNK1R is a membrane receptor for the neurotransmitter substance P.Polymerase chain reaction (PCR) technique was utilized for the isolationof human NK1R cDNA. In the approach, the regions of rat NK1R Applicantsthought to be similar to human NK1R were identified, oligonucleotideprimers corresponding to those region were designed, PCR amplificationwas carried out to obtain part of the NK1R cDNA from human cells, andits DNA sequence was determined. The remaining part of the human NK1RcDNA was obtained from a human cDNA library utilizing the above sequenceinformation of human NK1R cDNA.

The complete sequence of the human NK1R cDNA was determined, and itsencoded protein sequence was deduced. Among other things, such sequenceinformation is useful in the process of developing novel substance Pantagonists.

Three heterologous expression systems were used to express the clonedhuman NK1R cDNA. The Xenopus oocyte expression enables one to determinethe biological function of human NK1R. The COS (a monkey kidney cellline) expression can be used to measure the ligand binding properties ofhuman NK1R. The CHO (a Chinese hamster ovarian cell line) stableexpression is suitable for natural product screen to identify potentialtherapeutic agent or other substances that bind to substance P receptor.This cell line can also be used as an assay kit for determining the bodyfluid concentration of substance P in arthritis patients.

Assay protocols use the heterologously expressed human NK1R fordetermination of the binding affinity and antagonistic activity ofsubstance P antagonists.

1) Isolation of human NK1R cDNA

To isolate the human NK1R cDNA in the absence of its sequenceinformation, we developed methods to obtain three separate butoverlapping cDNA clones in three steps. (i) We have adopted thehomologous cloning strategy (0hara et al., 1989, Proc. Nat. Acad. Sci.,86:5673-5677) to isolate cDNA clones encoding the central core region ofhuman NK1R, with the assumption that the human NK1R sequence is similarto the published sequence (Yokota et al., 1989, J. Biol. Chem.,264:17649-17652) of rat NK1R in certain areas where appropriate PCRprimers can be designed. Degenerate primers corresponding to the ratsequence were used in PCR amplification (Mullis and Faloona, 1987, Meth.Enzymol., 155:335) to obtain the cDNA encoding the central trnsmembranecore region of human NK1R from human mRNA. (ii) After determining thesequence of the core region in human NK1R, new primers corresponding tothe human sequence were designed and a second homologous PCRamplification was performed using the human primer in the core regionwith degenerate primers corresponding to the N-terminal sequence of ratNK1R. The cDNA encoding the N-terminal region of human NK1R was thusobtained from human mRNA and its sequence was determined. (iii) Ananchored PCR strategy was developed to isolate the cDNA encoding theC-terminal region of human NK1R, in which primers corresponding to thecore region of human NK1R were used in combination with a primercorresponding to the sequence of a cloning vector to obtain the cDNAfrom a human cDNA library.

To confirm the authenticity of the cDNA encoding human NK1R, anindependent PCR amplification was performed to obtain the full lengthcDNA in a single step using primers from the 5' and 3' untranslatedregions.

2) Expression of the cloned human NK1R

Three expression systems were developed for the cloned human NK1R. Antransient expression in Xenopus oocytes resulted from microinjection ofin vitro transcribed mRNA from the cloned cDNA (Xenopus Laevis fromXENOPUS ONE, Ann Arbor, Mich.). This system allows the measurement ofbiological effect of NK1R activation upon ligand binding. Anothertransient expression in COS (a monkey kidney cell line, ATCC CRL 1651,ATCC Rockville Md.) resulted from the transfection of the cloned cDNAunder the control of vital promoter into mammalian cells (e.g., COS).The transfected cells are suitable for determination binding affinity ofhuman NK1R for various ligands. Stable expression of human NK1R inmammalian cells (e.g., CHO, a Chinese hamster ovarian cell line, ATCCCRL 9096, ATCC Rockville Md.) was achieved after integration of thetransfected cDNA into the chromosomes of the host cells. These stablecell lines will constituently express the cloned human NK1R and can bepropagated infinitely. Therefore, stable expression system is veryuseful in large scale drug screen, and can be used to determinesubstance p concentration in the biopsy sample of patients.

To establish a stable cell line expressing the cloned human NK1R, thecDNA was subcloned into the vector pRcCMV (INVITROGEN).

The electrophysiological assay of human NK1R expressed in Xenopusoocytes was based on the fact that NK1R activates the phospholipase Cupon substance P binding, and phospholipase C in turn increases theintracellular calcium concentration through inositol trisphosphate (IP₃)and IP₃ -gated calcium channel on intracellular membranes. The calciumincrease activates calcium-gated chloride channels on plasma membraneswhich gives rise to a chloride current measurable by two electrodevoltage clamp.

The binding assay of human NK1R expressed in COS or CHO is based on theuse of ¹²⁵ I-substance P (¹²⁵ I-SP, from DU PONT, Boston, Mass.) as aradioactively labeled ligand which compete with unlabeled substance p orany other ligand for binding to the human NK1R. Monolayer cell cultureof COS or CHO was dissociated by the non-enzymatic solution (SPECIALTYMEDIA, Lavallette, N.J.) and resuspended in appropriate volume of thebinding buffer (50 mM Tris pH 7.5, 5 mMMnCl₂, 150 mM NaCl, 0.04 mg/mlbacitracin, 0.004 mg/ml leupeptin, 0.2 mg/ml BSA, 0.01 mMphosphoramidon) such that 200 ul of the cell suspension would give riseto about 10,000 cpm of specific ¹²⁵ I-SP binding (approximately 50,000to 200,000 cells).

The activation of phospholipase C by NK1R can also be measured in CHOcells by determining the accumulation of inositol monophosphate which isa degradation product of IP₃.

In addition to large scale drug screening using the stable CHO cell lineexpressing the cloned human NK1R, other alternative applications areobvious. For example, the stable cell line can be used in the bindingassay to determine the substance p concentration from biopsy samples.The human NK1R protein can also be injected into patients to reducesubstance P concentration in some neurogenic inflammatory diseases.

EXAMPLE 1

Step A:

In the first step of obtaining the cDNA encoding the central core regionof human NK1R, human mRNA was prepared from three human glioblastomacell lines T98G, CCF-STTG1 and U87MG (obtained from the American TypeCulture Collection, Rockville, Md.) by the FASTTRACK method (INVITROGEN,San Diego, Calif.). Synthesis of first strand cDNA from 4 ug of humanmRNA was initiated by oligo (dT) primers in a total volume of 20 ulaccording to protocols of the BRL cDNA synthesis system (BRL, LIFETECHNOLOGIES, Inc., Gaithersburg, Md.). Ten ul of the first strand cDNAwas used as template with three rat primers (50 pmol rspr2s4, 50 pmolrspr2s4h, and 100 pmol rspr7a2; see Table I for their sequences) in aprimary PCR amplification in a total volume of 100 ul according to theGENEAMP protocol (PERKIN ELMER CETUS, Norwalk, Conn.). Thirty cycles ofPCR were performed using the following parameters: 1 min of denaturationat 94° C., 2 min of annealing at 40° C. and 4 min of extension at 72° C.with 2 sec of auto extension. Ten ul of the primary PCR product was usedas template with the same primers in a secondary PCR amplification underthe same cycling conditions to further amplify the DNA. Ten ul of thesecondary PCR product was used as template with three rat primers (50pmol rspr2s4, 50 pmol rspr2s4h, 50 pmol rspr7al and 50 pmol rspr7alh) in30 cycles of tertiary PCR amplification with the following parameters: 1min of denaturation at 94° C., 2 min of-annealing at 45° C., and 4 minof extension at 72° C. with 2 sec of auto extension. The tertiary PCRproduct was analyzed by agarose gel electrophoresis and was found tocontain a 600 bp DNA fragment. This DNA fragment was excised from thegel, purified by GENECLEAN (Bio 101, La Jolla, Calif.), phosphorylated,and subcloned into Sma I site of the plasmid vector BLUESCRIPT SK+(STRATAGENE, La Jolla, Calif.). The DNA sequence was determined by theSequenase dideoxy chain termination method (USBC, Cleveland, Ohio).Sequence alignment analysis showed that this cDNA fragment is similar(90% identity at nucleotide level) to the central core region of ratNK1R from amino acid 91 to 280.

STEP B:

After determination of the core region sequence of human NK1R, fiveantisense primers were synthesized based on the human sequence (hspr3a5,hspr5al, hspr5a2, hspr6al and hspr6a2; see Table II for theirsequences). These primers would be used to obtain the N-terminal cDNAsequence of human NK1R. One ug of human glioblastoma mRNA and 6 uM ofeach of the above primers was used in first strand cDNA synthesis in atotal volume of 20 ul according the BRL cDNA synthesis protocols. ThecDNA was extracted by phenol-chloroform, precipitated by ethanol anddissolved in 30 ul of water. Ten ul of the cDNA was used as templatewith two rat primers (50 pmol rsprn and 50 pmol rsprnh) and one humanprimer (150 pmol hspr3a5) in the primary PCR amplification in a totalvolume of 100 ul. Thirty cycles were performed with the followingparameters: 1 min denaturation at 94° C., 1 min of annealing at 55° C.,and 3 min of extension at 72° C. Five ul of the primary PCR product wasthen used as template with two rat primers (50 pmol rsprn and 50 pmolrsprnh) and one human primer (100 pmol hspr3a4) in 30 cycles ofsecondary PCR amplification with the same parameters. Two ul of thesecondary PCR product was used as template with two rat primers (50 pmolrsprn and 50 pmol rsprnh) and one human primer in 30 cycles of tertiaryPCR amplification with the same parameters. The tertiary PCR product wasanalyzed by agarose gel electrophoresis and was found to contain a 500bp fragment. This DNA fragment can hybridize with a humanoligonucleotide (hspr3a2), indicating it is not a non-specificby-product. This DNA fragment was excised from the gel, purified byGENECLEAN (Bio 101), phosphorylated, and subcloned into Sma I site ofthe vector Bluescript SK+. DNA sequence analysis revealed that thisfragment encodes the human NK1R N-terminal region and it also contains5' untranslated sequence.

STEP C:

In the third step, an anchored PCR protocol was developed in which thecDNA encoding the C-terminal region of human NK1R was obtained from acDNA library using sense human primers and a primer corresponding to thevector sequence. Three ug of human glioblastoma mRNA was primed by 2.5ug of oligo (dT) in the first strand cDNA synthesis in a total volume of50 ul, followed by second strand cDNA synthesis according the BRL cDNAsynthesis protocols. The cDNA product was then heated at 70° C. for 10min. The yield of double stranded cDNA was determined by incorporating1.25 uM of ³² P-a-dCTP as tracer in the reaction. Four ul of T4 DNApolymerase was added to the reaction mixture and incubated at 37° C. for10 min. The reaction was stopped by adding 16 ul of 250 mM EDTA,extracting with phenol/CHCl₃, and precipitating with ethanol. The cDNAwas dissolved in 50 ul of HE buffer (10 mM HEPES-lmM EDTA). Small sizecDNA was removed by the Select-D(RF) SPIN COLUMN (5'TO3', Boulder,Colo.), and the large size cDNA was precipitated by ethanol anddissolved in 36 ul of water. Four ul of 0.2M Tris-10 mM spermidine-1 mMEDTA (pH7.5) was added to the tube and heated at 70° C. for 1 min. ThecDNA was phosphorylated by adding 5 ul of blunt-end kinase buffer (0.5MTris pH 9.5, 0.1M MgCl₂, 50 mM DTT, 50% glycerol), 2.5 ul of 10 mMATP,2.5 ul of polynucleotide kinase, and incubating at 37° C. for 30 min.The cDNA was extracted by phenol/CHCl₃, precipitated by ethanol andligated to EcoRI linker according to the PROMEGA ECORI linker ligationprotocol (PROMEGA, Madison, Wis.). Linker-ligated cDNA was then ligatedto calf intestinal phosphatase-treated EcoRI site of the vectorBLUESCRIPT SK+. One ul of the ligated plasmid DNA was used as templatein 30 cycles of primary PCR with two human primers (50 pmol hspr6s1 and50 pmol hspr6s2) and 100 pmol of vector-specific primer t3 (obtainedfrom STRATAGEE) with the following parameters: 1 min of denaturation at94° C., 2 min of annealing at 55° C., and 4 min of extension at 72° C.with 2 sec auto extension. One ul of the primary PCR product was used in30 cycles of secondary PCR amplification with one human primer (100 pmolhspr6s3) and the same vector-specific primer t3 under the sameconditions. One ul of the secondary PCR product was used in 30 cycles oftertiary PCR amplification with one human primer (100 pmol hspr6s4) and100 pmol of vector-specific primer SK (STRATAGEE) under the sameconditions. A 780 bp DNA fragment was detected which also hybridized toa human oligo probe hspr6s5. This DNA fragment was excised from theagarose gel, purified by GENECLEAN (BIO 101), phosphorylated, andsubcloned into Sma I site of the vector BLUESCRIPT SK+. DNA sequenceanalysis revealed that it encodes the C-terminal region of human NK1Rand contains 3' untranslated sequence.

STEP D:

Since three separate but overlapping cDNA clones encoding human NK1Rwere isolated above and the possibility of alternative pre-mRNA splicingexists, it is necessary to confirm the authenticity of the full lengthcDNA sequence by isolating a full length cDNA directly. Based on theabove sequence in the untranslated region, primers were synthesizedwhich should give rise to a full length cDNA. Using the PERKIN ELMERCETUS RNA PCR amplification kit (Perkin Elmer Cetus), cDNA wassynthesized from 1.5 ug of human glioblastoma mRNA in a total volume of20 ul with 50 pmol of the human primer hspr3uta5. One half of the firststrand cDNA was used as template in 30 cycles of primary PCRamplification with two human primers (50 pmol hspr3uta5, 50 pmolhspr5utsl) with the following parameters: 1 min of denaturation at 94°C., 2 min of annealing at 55° C., and 4 min of extension at 55° C. with2 sec auto extension. Ten ul of the primary PCR product was used astemplate in 30 cycles of secondary PCR amplification with two humanprimers (50 pmol hspr3uta6 and 50 pmol hspr5uts2) under the sameconditions. A 1350 bp DNA fragment was excised from agarose gel,purified by GENECLEAN (BIO 101), digested with restriction endonucleaseswith EcoRI and Not I, and subcloned into the vector BLUESCRIPT SK+. DNAsequence analysis confirmed the general structure of the cloned humanNK1R cDNA. The sequence of human NK1R CDNA is shown in FIG. 2.

    __________________________________________________________________________    Name  Sequence (SEQ ID NO:.sub.-- :)                                                                      Position                                                                            Direction                                   __________________________________________________________________________    rspr2s4                                                                             TGCATGGCTGCATTCAAT (2)                                                                              238-255                                                                             sense                                       rspr2s4h                                                                            TGCATGGCTGCCTTCAA (3) 238-254                                                                             sense                                       rspr7a2                                                                             ACAGTAGATGATGGGGTTGTACAT (4)                                                                        918-894                                                                             antisense                                   rspr7a1                                                                             CAGGTAGACCTGCTGGATGAACTT (5)                                                                        864-841                                                                             antisense                                   rspr7a1h                                                                            CAGGTACACCTGCTGGATGAACTT (6)                                                                        864-841                                                                             antisense                                   rsprn ATGGATAACGTCCTTCCTAT (7)                                                                             1-20 sense                                       rsprnh                                                                              ATGGACAATGTGCTGCCCA (8)                                                                              1-19 sense                                       __________________________________________________________________________

    __________________________________________________________________________    Name  Sequence (SEQ ID NO:.sub.-- :)                                                                             Position                                                                            Direction                            __________________________________________________________________________    hspr3a2                                                                             GAAGAAGTTGTGGAACTTGCA (9)    455-435                                                                             antisense                            hspr3a1                                                                             CATGGAGTAGATACTGGCGAA (10)   491-471                                                                             antisense                            hspr3a4                                                                             GGATGTATGATGGCCATGTA (11)    532-513                                                                             antisense                            hspr3a5                                                                             ACTTTGGTGGCTGTGGCTGA (12)    568-549                                                                             antisense                            hspr5a1                                                                             ATGCATAGCCAATCACCAGCA (13)   768-748                                                                             antisense                            hspr5a2                                                                             CATAGTGTGATTCCCACTAC (14)    793-774                                                                             antisense                            hspr6a1                                                                             TGCACACCACGACAATCATCA (15)   888-868                                                                             antisense                            hspr6a2                                                                             TTGATGTAGGGCAGGAGGAA (16)    943-924                                                                             antisense                            hspr6s1                                                                             GCAAGTCTCTGCCAAGCGCAA (17)   836-856                                                                             sense                                hspr6s2                                                                             TGATGATTGTCGTGGTGTGCA (18)   868-888                                                                             sense                                hspr6s3                                                                             TTCCACATCTTCTTCCTCCT (19)    912-931                                                                             sense                                hspr6s4                                                                             CTACATCAACCCAGATCTCT (20)    935-954                                                                             sense                                hspr6s5                                                                             TCTCTACCTGAAGAAGTT (21)      950-967                                                                             sense                                hspr12uta1                                                                          CAAGGATGGAATGTTTTCCCT (22)   1499-1479                                                                           antisense                            hspr12uta2                                                                          (GACATGCGGCCGC)AACCCATACTGACCCTTTT(23)                                                                     1478-1460                                                                           antisense                            hspr5uts1                                                                           CCTCCTGTCTGGCTTTAGAA (24)    16-35 sense                                hspr5uts2                                                                           (GCGCAGAATTC)GTGTACAGATAGTAGGCTT (25)                                                                       86-105                                                                             sense                                __________________________________________________________________________

Expression in Xenopus oocytes

To express the human NK1R cDNA in Xenopus oocytes, the cDNA was clonedinto an in vitro transcription vector BLUESCRIPT SK+ (STRATAGENE) whichcontains the T7 promoter for initiation of T7 RNA polymerase catalyzedRNA synthesis. One ug of linear plasmid DNA which contained the humanNK1R cDNA downstream of the T7 promoter was used in the in vitrotranscription reaction containing 40 mM Tris pH 7.5, 50 mM NaCl, 8 mMMgCl₂, 2 mM spermidine, 0.4 mM CTP, 0.4 mM ATP, 0.4 mM UTP, 0.16 mM GTP,2.5 ul CAP analog (STRATAGENE), 30 mM DTT, 1 U RNase Block II(STRATAGENE), 0.83 pmol ³² P-a-CTP and 25 U of T7 RNA polymerase. Thereaction tube was incubated at 37° C. for 1 hour. Usually 5 ug of RNAwas synthesized as quantitated by incorporation of ³² P-a-CTP into RNA.After RNA synthesis, the plasmid DNA was removed by adding 10 U of RNasefree DNase and 1 U of RNase Block II. The reaction mixture was extractedby phenol/CHCl₃, and the unincorporated nucleotides were removed by theSelect-D(RF) spin column (5'T03'). The RNA transcript was precipitatedby ethanol twice and dissolved in RNase free water. Oocytes were removedfrom Xenopus frogs, treated with 2 mg/ml collagenase (specificactivity<0.3 U/mg, BOEHRINGER MANNHEIM, Indianapolis, Ill.) in 0R-2buffer (82.5 mM NaCl, 2 mM KCl, 1 mMMECl₂, 5 mMHEPES, pH 7.4) for 4hours at 19° C. The dissociated oocytes were incubated in OR-2 buffersupplemented by 1.8 mM CaCl₂, 0.5 mg/ml gentamycin and 0.5 mMtheophylline at 19° C. overnight before injection. A 50 nl aliquotcontain 2 ng of RNA transcript was injected into each oocyte. Theinjected oocytes Were incubated at 19° C. for 2 days beforeelectrophysiological recording (see Example 3 for assay method).

Expression in COS

To express the human NK1R transiently in COS, the cDNA was cloned intothe expression vector pCDM9 which was derived from pCDM8 (INVITROGEN) byinserting the ampicillin resistance gene (nucleotide 1973 to 2964 fromBLUESCRIPT SK+) into the Sac II site. Transfection of 20 ug of theplasmid DNA into 10 millions COS cells was achieved by electropotationin 800 ul of transfection buffer (135 mM NaCl, 1.2 mM CaCl₂, 1.2 mMMgCl₂, 2.4 mM K₂ HPO₄, 0.6 mM KH₂ PO₄, 10 mM glucose, 10 mM HEPES pH7.4) at 260 V and 950 uF using the IBI GENEZAPPER (IBI, New Haven,Conn.). The cells were incubated in 10% fetal calf serum, 2 mMglutamine, 100U/ml penicillin-streptomycin, and 90% DMEM media (GIBCO,Grand Island, N.Y.) in 5% CO₂ at 37° C. for three days before thebinding assay.

Stable Expression in CHO

To establish a stable cell line expressing the cloned human NK1R, thecDNA was subcloned into the vector pRcCMV (INVITROGEN). Transfection of20 ug of the plasmid DNA into CHO cells was achieved by electropotationin 800 ul of transfection buffer suplemented with 0.625 mg/ml Herringsperm DNA at 300 V and 950 uF using the IBI GENEZAPPER (IBI). Thetransfected cells were incubated in CHO media [10% fetal calf serum, 100U/ml pennicilin-streptomycin, 2 mM glutamine, 1/500hypoxanthine-thymidine (ATCC), 90% IMDM media (3RH BIOSCIENCES, Lenexa,Kans.), 0.7 mg/ml G418 (GIBCO)] in 5% CO₂ at 37° C. until colonies werevisible. Each colony was separated and propagated. The cell clone withthe highest number of human NK1R was selected for subsequent applicationin the assay of Example 3.

EXAMPLE 3

Assay Protocol Using Oocytes

The oocyte was voltage-clamped at -80 mV by the model 8500 intracellularpreamp-clamp (DAGAN, Minneapolis, Minn.). The recoding chamber wascontinuously perfused with recording buffer (96 mM NaCl₁, 2 mM KCl, 1.8mM CaCl₂, 5 mM HEPES, pH 7.4). Chloride current was elicited by applyingsubstance P (from 0.1 nM to 1000 nM) to the recording chamber. At leastthree oocytes were measured for each concentration. The antagonisticactivity of any potential substance P antagonist can be assessed bydetermining the inhibition of substance P response. Likewise, NK1agonists can be identified by their ability to stimulate a response inoocytes injected with NK1R mRNA but not in uninjected oocytes.

Assay Protocol using COS or CHO

The binding assay of human NK1R expressed in COS or CHO is based on theuse of ¹²⁵ I-substance P (¹²⁵ I-SP, from DU PONT, Boston, Mass.) as aradioactively labeled ligand which compete with unlabeled substance p orany other ligand for binding to the human NK1R. Monolayer cell cultureof COS or CHO was dissociated by the non-enzymatic solution (SPECIALTYMEDIA, Lavallette, N.J.) and resuspended in appropriate volume of thebinding buffer (50 mM Tris pH 7.5, 5 mMMnCl₂, 150 mM NaCl, 0.04 mg/mlbacitracin, 0.004 mg/ml leupeptin, 0.2 mg/ml BSA, 0.01 mMphosphoramidon) such that 200 ul of the cell suspension would give riseto about 10,000 cpm of specific ¹²⁵ I-SP binding (approximately 50,000to 200,000 cells). In the binding assay, 200 ul of cells were added to atube containing 20 ul of 1.5 to 2.5 nM of ¹²⁵ I-SP and 20 11 ofunlabeled substance p or any other test compound. The tubes wereincubated at 4° C. or at room temperature for 1 hour with gentleshaking. The bound radioactivity was separated from unboundradioactivity by GF/C filter (BRANDEL, Gaithersburg, Md.) which waspre-wetted with 0.1% polyethylenimine. The filter was washed with 3 mlof wash buffer (50 mM Tris pH 7.5, 5 mM MnCl₂, 150 mM NaCl) three timesand its radioactivity was determined by gamma counter. Illustrative ofthis method of using these expression systems are the results shoen inFIG. 3. These results show the competitive binding of substance P (SP),substance K (SK) and human neurokinin-1 receptor (NK1R) in the COSassay.

ALTERNATIVE PROTOCOL

The activation of phospholipase C by NK1R can also be measured in CHOcells by determining the accumulation of inositol monophosphate which isa degradation product of IP₃. CHO cells were seeded in 12-well plate at250,000 cells per well. After incubating in CHO media for 4 days, cellswere loaded with 0.025 uCi/ml of ³ H-myoinositol by overnightincubation. The extracellular radioactivity was removed by washing withphosphate buffered saline. LiCl was added to the well at finalconcentration of 0.1 mM with or without antagonist, and continuedincubation at 37° C. for 15 min. Substance p was added to the well atfinal concentration of 0.3 nM to activate the human NK1R. After 30 minof incubation at 37° C., the media was removed and 0.1N HCl was added.Each well was sonicated at 4° C. and extracted with CHCl₃ /methanol(1:1). The aqueous phase was applied to a 1 ml Dowex AG 1X8 ion exchangecolumn. The column was washed with 0.1N formic acid followed by 0.025Mammonium formate-0.1N formic acid. The inositol monophosphate was elutedwith 0.2M ammonium formate-0.1N formic acid and quantitated by betacounter.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 27                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 122 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAAAAAGCCTTCCACCCTCCTGTCTGGCTTTAGAAGGACCCTGAGCCCCA50                          GGCGCCACGACAGGACTCTGCTGCAGAGGGGGGTTGTGTACAGATAGTAG100                         GGCTTTACCGCCTAGCTTCGAA 122                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TGCATGGCTGCATTCAAT 18                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TGCATGGCTGCCTTCAA 17                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ACAGTAGATGATGGG GTTGTACAT24                                                   (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAGGTAG ACCTGCTGGATGAACTT24                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAGGTACACCTGCTGGATGAACTT24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (x i) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                      ATGGATAACGTCCTTCCTAT20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGGACAATGTGCTGCCCA19                                                         (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE:                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GAAGAAGTTGTGGAACTTGCA21                                                       (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CATGGAGTAGATACTGGCGAA21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGATGTATGATGGCCATGTA20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C ) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ACTTTGGTGGCTGTGGCTGA20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ATGCATAGCCAATCACCAGCA21                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CATAGTGTGATTCCCACTAC20                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TGCACACCACGACAATCATCA21                                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TTGATGTAGGGCAGGAGGAA20                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCAAGTCTCTGCCAAGCGCAA21                                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TGATGATTGTCGTGGTGTGCA21                                                       (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TTCCACATCTTCTTCCTCCT20                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CTACATCAACCCAGATCTCT20                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TCTCTACCTGAAGAAGTT 18                                                         (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CAAGGATGGAATGTTTTCCCT 21                                                      (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AACCCATACTGACCCTTTT 19                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CCTCCTGTCTGGCTTTAGAA 20                                                       (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GTGTACAGATAGTAGGCTT 19                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 407 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      MetAspAsnV alLeuProValAspSerAspLeuSerProAsnIleSer                             151015                                                                        ThrAsnThrSerGluProAsnGlnPheValGlnProAlaTrpGlnIle                               202530                                                                       ValLeuTrpAlaAlaAlaTyrThrValIleValValThrSerValVal                              354045                                                                        GlyAsnVal ValValMetTrpIleIleLeuAlaHisLysArgMetArg                             505560                                                                        ThrValThrAsnTyrPheLeuValAsnLeuAlaPheAlaGluAlaSer                              65 707580                                                                     MetAlaAlaPheAsnThrValValAsnPheThrTyrAlaValHisAsn                              859095                                                                        GluT rpTyrTyrGlyLeuPheTyrCysLysPheHisAsnPhePhePro                             100105110                                                                     IleAlaAlaValPheAlaSerIleTyrSerMetThrAlaValAlaPhe                               115120125                                                                    AspArgTyrMetAlaIleIleHisProLeuGlnProArgLeuSerAla                              130135140                                                                     ThrAlaThr LysValValIleCysValIleTrpValLeuAlaLeuLeu                             145150155160                                                                  LeuAlaPheProGlnGlyTyrTyrSerThrThrGluThrMetProSer                               165170175                                                                    ArgValValCysMetIleGluTrpProGluHisProAsnLysIleTyr                              180185190                                                                      GluLysValTyrHisIleCysValThrValLeuIleTyrPheLeuPro                             195200205                                                                     LeuLeuValIleGlyTyrAlaTyrThrValValGlyIleThrLeuTrp                              210215220                                                                     AlaSerGluIleProGlyAspSerSerAspArgTyrHisGluGlnVal                              225230235240                                                                   SerAlaLysArgLysValValLysMetMetIleValValValCysThr                             245250255                                                                     PheAlaIleCysTrpLeuProPheHisIlePhePheLeuLeu ProTyr                             260265270                                                                     IleAsnProAspLeuTyrLeuLysLysPheIleGlnGlnValTyrLeu                              2752802 85                                                                    AlaIleMetTrpLeuAlaMetSerSerThrMetTyrAsnProIleIle                              290295300                                                                     TyrCysCysLeuAsnAspArgPheArgLeuGlyPheLysHisAlaPh e                             305310315320                                                                  ArgCysCysProPheIleSerAlaGlyAspTyrGluGlyLeuGluMet                              325330 335                                                                    LysSerThrArgTyrLeuGlnThrGlnGlySerValTyrLysValSer                              340345350                                                                     ArgLeuGluThrThrIleSerThrValValGlyA laHisGluGluGlu                             355360365                                                                     ProGluAspGlyProLysAlaThrProSerSerLeuAspLeuThrSer                              370375 380                                                                    AsnCysSerSerArgSerAspSerLysThrMetThrGluSerPheSer                              385390395400                                                                  PheSerSerAsnValLeuSer                                                          405                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1679 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GAAAAAGCCTTCCACCCTCCTGTCTGGCTTTAGAAGGACCCTGAGCCCC AGGCGCCACGA60               CAGGACTCTGCTGCAGAGGGGGGTTGTGTACAGATAGTAGGGCTTTACCGCCTAGCTTCG120               AAATGGATAACGTCCTCCCGGTGGACTCAGACCTCTCCCCAAACATCTCCACTAACACCT180               CGGAACCCAATCAGTTCGTGCAA CCAGCCTGGCAAATTGTCCTTTGGGCAGCTGCCTACA240              CGGTCATTGTGGTGACCTCTGTGGTGGGCAACGTGGTAGTGATGTGGATCATCTTAGCCC300               ACAAAAGAATGAGGACAGTGACGAACTATTTTCTGGTGAACCTGGCCTTCGCGGAGGCCT360               CCATGGCTGCATTCAATACAGTGGTGAACTTCACCTATGCTGTCCACAACGAATGGTACT420               ACGGCCTGTTCTACTGCAAGTTCCACAACTTCTTCCCCATCGCCGCTGTCTTCGCCAGTA480               TCTACTCCATGACGGCTGTGGCCTTTGATAGGTACATGGCC ATCATACATCCCCTCCAGC540              CCCGGCTGTCAGCCACAGCCACCAAAGTGGTCATCTGTGTCATCTGGGTCCTGGCTCTCC600               TGCTGGCCTTCCCCCAGGGCTACTACTCAACCACAGAGACCATGCCCAGCAGAGTCGTGT660               GCATGATCGAATGGCC AGAGCATCCGAACAAGATTTATGAGAAAGTGTACCACATCTGTG720              TGACTGTGCTGATCTACTTCCTCCCCCTGCTGGTGATTGGCTATGCATACACCGTAGTGG780               GAATCACACTATGGGCCAGTGAGATCCCCGGGGACTCCTCTGACCGCTACCACGAGCAAG 840              TCTCTGCCAAGCGCAAGGTGGTCAAAATGATGATTGTCGTGGTGTGCACCTTCGCCATCT900               GCTGGCTGCCCTTCCACATCTTCTTCCTCCTGCCCTACATCAACCCAGATCTCTACCTGA960               AGAAGTTTATCCAGCAGGTCTACCTGGCCATCAT GTGGCTGGCCATGAGCTCCACCATGT1020             ACAACCCCATCATCTACTGCTGCCTCAATGACAGGTTCCGTCTGGGCTTCAAGCATGCCT1080              TCCGGTGCTGCCCCTTCATCAGCGCCGGCGACTATGAGGGGCTGGAAATGAAATCCACCC1140              GGTATCTCC AGACCCAGGGCAGTGTGTACAAAGTCAGCCGCCTGGAGACCACCATCTCCA1200             CAGTGGTGGGGGCCCACGAGGAGGAGCCAGAGGACGGCCCCAAGGCCACACCCTCGTCCC1260              TGGACCTGACCTCCAACTGCTCTTCACGAAGTGACTCCAAGACCATGACAGA GAGCTTCA1320             GCTTCTCCTCCAATGTGCTCTCCTAGGCCACAGGGCCTTTGGCAGGTGCAGCCCCCACTG1380              CCTTTGACCTGCCTCCCTTCATGCATGGAAATTCCCTTCATCTGGAACCATCAGAAACAC1440              CCTCACACTGGGACTTGCAAAAAGGGT CAGTATGGGTTAGGGAAAACATTCCATCCTTGA1500             GTCAAAAAATCTCAATTCTTCCCTATCTTTGCCACCCTCATGCTGTGTGACTCAAACCAA1560              ATCACTGAACTTTGCTGAGCCTGTAAAATAAAAGGTCGGACCAGCTTTTCCCAAAAGCCC1620              A TTCATTCCATTCTGGAAGTGACTTTGGCTGCATGCGAGTGCTCATTTCAGGATGAATT1679          

What is claimed is:
 1. A recombinant protein having the amino acidsequence SEQ ID NO:26 of the human neurokinin-1 receptor.